Influence of pre-analytical conditions on the measurement of components of the GH/IGF axis in rats

Pre-analytical variability characterises effects which are introduced to an analysis by manipulation and storage of a biological sample after taking it ex-vivo, but before actually quantifying the respective analyte. In humans, recent studies demonstrated that pre-analytical factors can be an important confounder for immunoassay measurements of circulating hormones of the GH/IGF-system. In contrast, this topic has rarely been addressed in rodent studies. We therefore now systematically investigated if and how commonly used sample processing and pre-treatment protocols affect measured hormone concentrations of the GH/IGF system in rats. Furthermore, we explored if short term fasting, age and circadian rhythmicity have an impact upon the concentrations of IGF-I, IGFBP-2 and IGFBP-3 in rats.ResultsOn average, concentrations of IGF-I were lower by 9.2% (p < 0.01), while those of IGF-II and IGFBP-3 were lower by 24% (p < 0.01) in EDTA plasma when compared to plain serum from the same rats. In contrast, concentrations of GH were significantly higher in plain plasma samples compared with serum (+ 137.8%; p < 0.01). Repeated freeze/thaw cycles significantly influenced the measurements of serum IGF-II (+ 25.9%; p < 0.01) and IGFBP-3 (+ 19.3%; p < 0.01) when compared to native serum. Pre-treatment of EDTA plasma with protease inhibitors, or immediate storage of EDTA blood on ice, did not significantly affect the outcome of any measurement. Acidification of plasma samples with HCl led to significantly lower IGF-I in samples (? 11.9%, p < 0.001) and detection of GH was completely hampered in these samples. With respect to biological variability, age (12-week-old vs. 1-year-old male Wistar rats), but not fasting (up to 18 h) or circadian rhythmicity affected circulating concentrations of IGF-I and IGFBP-3.ConclusionPre-analytical variability is a potentially confounding factor which also must be considered in rodent studies when analysing and comparing hormones of the GH/IGF system. If and to what extent a specific pre-analytical procedure affects immunoassay measurements in rodent studies cannot be predicted in advance but rather needs to be tested for each analyte separately.     

Leptin concentrations in GH deficiency: the effect of GH insensitivity

Disorders of GH secretion are known to impair the physiological lipostat and to affect the secretion of leptin, a sensitive marker of regional fat accumulation and total body composition. In both children and adults with GH deficiency (GHD), leptin levels are increased proportionately with enhanced adiposity. In GHI, mutations of the GH receptor gene result in a phenotype similar to GHD, with increased adiposity and unfavorable lipid profiles. To examine the impact of different forms of growth disorders on leptin production, we measured leptin levels in 22 GHI patients homozygous for the E180 splice mutation (15 females and 7 males, aged 8-37 yr) and compared results with those obtained in 20 subjects heterozygous for the mutation (11 females and 9 males, aged 7-54), 17 idiopathic GHD patients (6 females and 11 males, aged 3-34), and 44 normal subjects (25 females and 19 males, aged 7-45). After the baseline evaluation, all subjects received two 7-d GH treatments at doses of 0.025 and 0.050 mg/kg x d in random order. Leptin, IGF-I, and IGF-binding protein-3 (IGFBP-3) were assayed by specific immunoassays. IGF-I and IGFBP-3 levels were significantly lower (P < 0.0001) in homozygous GHI and GHD patients compared with either controls or GHI heterozygotes. Circulating leptin levels were significantly higher in homozygous GHI patients than in normal controls (20.7 +/- 4.2 vs. 8.7 +/- 1.4 microg/liter) as well as when compared with heterozygous GHI subjects (14.4 +/- 3.4 microg/liter) and GHD patients (9.8 +/- 1.6 microg/liter; P < 0.01). Similar results were obtained when leptin was normalized for body mass index. When subjects were subgrouped by gender, leptin levels were significantly higher (P < 0.05) in GHI females than in females of all other groups and were significantly increased in GHD males (P < 0.01 vs. control males). Within the study groups, females had significantly higher leptin levels than males in controls (12.7 +/- 2 vs. 3.3 +/- 1 microg/liter; P < 0.001) and homozygous GHI patients (28.7 +/- 5.3 vs. 6.9 +/- 2.3 microg/liter; P < 0.05), but not in heterozygous GHI (20.1 +/- 5.4 vs. 7.3 +/- 2.4 microg/liter; P < 0.06) and GHD (10.9 +/- 2.6 vs. 9.2 +/- 2.1 microg/liter) patients. By multivariate analysis, log-normalized leptin levels were best predicted by gender and body mass index in homozygous GHI patients as well as in normal subjects. During the 1-wk courses of GH therapy, serum IGF-I and IGFBP-3 levels significantly increased (P < 0.0001) in GHD patients, heterozygous GHI patients, and control subjects at both GH doses. Inversely, leptin levels did not change significantly during either course of GH administration in the groups examined. These data demonstrate that leptin is increased in patients affected with long-standing homozygous GHI, probably reflecting abnormalities of body composition and metabolism typical of this condition.     

Central reassessment of GH concentrations measured at local treatment centers in children with impaired growth: consequences for patient management

OBJECTIVE: GH deficiency is diagnosed in children if serum GH fails to rise above a predefined cutoff value in response to at least two stimuli. Diagnostic decisions based on this testing are highly variable between centers and depend on the GH assays used. Considering the large spectrum of commercially available GH assays, we wanted to evaluate the agreement between assays, and to test whether assay-related variability of diagnostic decisions could be reduced by reassessment of peak GH concentrations in a reference center. DESIGN: We reanalysed 699 peak GH serum samples obtained after GH testing of 382 children and adolescents from 19 centers using three reference assays and compared these results with those obtained with the local assays. A subgroup of 132 patients tested with the combination of insulin hypoglycemia test and arginine test was evaluated for changes in the assignment to the diagnostic group of GH deficiency. RESULTS: The mean difference between methods ranged from 5.4 to 10.3 mU/l, slopes of the regression lines from 1.28 to 1.65. Significant non-linearity was detected in five of six assay comparisons, indicating that most assay results cannot be interconverted by the use of a factor. Overall agreement between reference and local assays was only moderate. Significant changes in diagnostic assignment occurred when different assays were used on the same patient (P<0.0001-P<0.0023). Based on GH remeasurement by one reference assay, 36 of 132 patients were categorized differently, with 35 patients changing into the GH-deficient group. Similar findings were obtained with the other reference assays. CONCLUSIONS: To decrease variability in GH testing related to assays and cutoff values, we recommend nationwide reassessment of GH peak sera in reference centers. Decisions to treat GH deficiency should incorporate the reference center results.     

Effects of the IGF-I/IGFBP-3 complex on GH and ghrelin nocturnal concentrations in low birth weight children

OBJECTIVE: There is limited information regarding the effects of IGF-I and/or IGFBP-3 on circulating ghrelin concentrations. To determine the effects of IGF-I on GH and ghrelin concentrations, we examined the GH and ghrelin nocturnal profiles before and after the administration of the IGF-I/-IGFBP-3 complex (Iplex) to low birth weight children. DESIGN: The children were studied on two separate occasions, the first under basal conditions, and the second time after the sc administration of 1 mg/kg of Iplex at 2100 h. Blood samples for determination of GH and ghrelin were obtained every 20 min between 2300 h and 0700 h, while the children were sleeping. In each patient, we calculated the mean GH and ghrelin area under the curve (GH AUC and GHR AUC), both under basal conditions and after the administration of the IGF-I/IGFBP-3 complex. SETTING: The study was performed at a University Research Centre located at a General Hospital in Santiago, Chile. PATIENTS: Twenty prepubertal children (11 boys and 9 girls), born after a full-term pregnancy with a birth weight below 2.8 kg were studied at a mean +/- SEM age of 7.3 +/- 0.5 years (range 4-11 years). Their mean height was -1.8 +/- 0.3 standard deviation score (SDS) and their mean BMI was 0.1 +/- 0.2 SDS at the time of the study. MAIN OUTCOME AND RESULTS: Mean nocturnal GH AUC exhibited a significant decrease (2903 +/- 185 vs 1860 +/- 122 ng/ml min, P < 0.01), whereas mean GHR AUC showed a significant increase after administration of the IGF-I/IGFBP-3 complex (68 +/- 16 vs 288 +/- 36 ng/ml min, P < 0.01). CONCLUSIONS: These findings indicate that the IGF-I/IGFBP-3 complex appears to have opposite effects on circulating GH and ghrelin concentrations in low birth weight children, suggesting that, in addition to its known negative feed-back effect on GH, IGF-I and/or IGFBP-3 may have a positive feed-back effect on ghrelin.     

Activation of somatostatin-receptor subtype-2/-5 suppresses the mass, frequency, and irregularity of growth hormone (GH)-releasing peptide-2-stimulated GH secretion in men

Somatostatin antagonizes the stimulatory actions of GHRH and GH-releasing peptides (GHRPs). However, precisely how the inhibitory susceptibilities of the two secretagogues differ is not clear. One interpretative difficulty is that native somatostatin activates six different receptor subtypes. The present study adopts the complementary strategy of enforcing feedback inhibition via the preferential somatostatin receptor subtype 2 and 5 (SSTR-2/-5) agonist, octreotide. We postulated that putative SSTR-2/-5 agonism would unmask secretagogue-selective interactions in the control of GH secretory burst mass, frequency, and/or regularity. To this end, 10 healthy men each underwent eight randomly ordered, separate-day, fasting morning infusion sessions. Interventions comprised sc administration of octreotide (1 microg/kg), followed by bolus iv injection of saline, GHRH (1 microg/kg), GHRP-2 (1 microg/kg), or both peptides. Compared with placebo, the SSTR-2/-5 agonist reduced fasting GH concentrations from 0.27 +/- 0.07 to 0.12 +/- 0.02 microg/liter (P = 0.020), GH secretory burst mass from 2.7 +/- 0.65 to 0.55 +/- 0.11 microg/liter (P = 0.013), and basal GH secretion from 0.24 +/- 0.043 to 0.11 +/- 0.015 microg/liter.100 min (P = 0.0063). The foregoing outcomes were selective, because octreotide did not alter GH secretory burst frequency (3.1 +/- 0.5 vs. 3.3 +/- 0.21 events/3 h) or the regularity of the GH release process (approximate entropy, 0.58 +/- 0.048 vs. 0.68 +/- 0.064). In the GHRP-2-stimulated setting, presumptive SSTR-2/-5 agonism suppressed all three GH secretory burst masses, from 28 +/- 3.2 to 18 +/- 2.0 (P = 0.045); GH pulse frequency, from 3.3 +/- 0.30 to 2.0 +/- 0.18 (P = 0.0025); and the irregularity (approximate entropy) of GH release, from 0.648 +/- 0.049 to 0.433 +/- 0.047 (P < 0.01). In contrast, in the GHRH and combined GHRH/GHRP-2-stimulated contexts, octreotide decreased only GH secretory burst mass (P = 0.047). In summary, the present data indicate that GH secretory burst mass, frequency, and orderliness are subject to interactive control by at least SSTR-2/-5-dependent feedback and GHRP-dependent feedforward signals.     

Effects of swimming training on bone mass and the GH/IGF-1 axis in diabetic rats

The aim of this study was to examine the influence of moderate swimming training on the GH/IGF-1 growth axis and tibial mass in diabetic rats. Male Wistar rats were allocated to one of four groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD) and trained diabetic (TD). Diabetes was induced with alloxan (35 mg/kg b.w.). The training program consisted of a 1h swimming session/day with a load corresponding to 5% of the b.w., five days/week for six weeks. At the end of the training period, the rats were sacrificed and blood was collected for quantification of the serum glucose, insulin, GH, and IGF-1 concentrations. Samples of skeletal muscle were used to quantify the IGF-1 peptide content. The tibias were collected to determine their total area, length and bone mineral content. The results were analyzed by ANOVA with P<0.05 indicating significance. Diabetes decreased the serum levels of GH and IGF-1, as well as the tibial length, total area and bone mineral content in the SD group (P<0.05). Physical training increased the serum IGF-1 level in the TC and TD groups when compared to the sedentary groups (SC and SD), and the tibial length, total area and bone mineral content were higher in the TD group than in the SD group (P<0.05). Exercise did not alter the level of IGF-1 in gastrocnemius muscle in nondiabetic rats, but the muscle IGF-1 content was higher in the TD group than in the SD group. These results indicate that swimming training stimulates bone mass and the GH/IGF-1 axis in diabetic rats.     

丹参素异丙酯对慢性缺氧大鼠血压和左心室功能的影响

目的: 研究丹参素异丙酯对缺氧大鼠左心室功能的影响。方法: 制备慢性缺氧大鼠模型,分别用大、小剂量的丹参素异丙酯灌胃,设立维拉帕米阳性对照组和正常对照组,采用大鼠心导管插入的方法,测定大鼠血流动力学和心功能指标,观察不同剂量丹参素异丙酯对慢性缺氧大鼠左室心功能的影响。结果: 与正常组比较,单纯模型组的动脉收缩压（SBP）、动脉舒张压（DBP）、平均动脉压（MBP）、左室收缩压（LVSP）、最大上升速率（+dp/dtmax）和最大下降速率（-dp/dtmax）显著升高（P<0.05或P<0.01）,T值(t-dp/dtmax)显著降低（P<0.05）,HE染色显示,心肌细胞出现肌纤维断裂、溶解,细胞核固缩、溶解。与单纯模型组比较,小剂量组的DBP、MBP、心率(HR)、左室舒张末压(LVEDP)和-dp/dtmax显著降低（P<0.05或P<0.01）,T值显著升高（P<0.05）;大剂量组的SBP、DBP、MBP、HR、LVEDP、左室舒张压(LVDP)、+dp/dtmax和-dp/dtmax显著降低（P<0.05或P<0.01）,T值显著升高（P<0.01）;HE染色显示,大、小剂量组心肌细胞内糖元含量丰富,肌纤维排列相对规则,未见明显的肌纤维断裂、溶解及核分裂。与阳性对照组比较,小剂量组的DBP、MBP、LVSP、LVDP和T值显著升高（P<0.05或P<0.01）,LVEDP显著降低（P<0.05）;大剂量组的DBP和T值均显著升高（P<0.05或P<0.01）,LVDP、LVEDP和+dp/dtmax均显著降低（P<0.05或P<0.01）。与小剂量组比较,大剂量组的LVDP、LVEDP和+dp/dtmax均显著降低（P<0.01）。结论: 丹参素异丙酯对慢性缺氧造成的大鼠血压升高、心率加快、心功能亢奋等有明显的抑制作用,因而对缺氧大鼠的心功能有明显的保护作用。     

Effect of restricted feeding on the relationship between hypophysial portal concentrations of growth hormone (GH)-releasing factor and somatostatin, and jugular concentrations of GH in ovariectomized ewes.

These studies characterized the secretion of GH-releasing factor (GRF) and somatostatin (SRIF) into the hypophysial portal circulation in ewes after long term restricted feeding. In addition, we examined the temporal relationship between the concentrations of these two hypothalamic peptides in portal blood and the concentration of GH in jugular blood. Six sheep were fed 1000 g hay/day (normal feeding) and 6 sheep were fed 400-600 g hay/day (restricted feeding). This resulted in a wt loss of 35% in restricted animals compared with 6% in control animals after 20 weeks. Fluctuations in portal levels of GRF indicated a pulsatile pattern of secretion with approximately 60% of pulses coincident with, or immediately preceding, a GH pulse. Similarly, 65% of GH pulses were associated with GRF pulses. Restricted feeding increased (P less than 0.01) mean ( +/- SEM) plasma GH levels (9.8 +/- 1.4 vs. 2.9 +/- 0.6 ng/ml) and mean GH pulse amplitude (7.9 +/- 1.8 vs. 2.8 +/- 0.3 ng/ml) but did not affect mean GH pulse frequency (6.0 +/- 1.1 vs. 5.7 +/- 1.1 pulses/8 h). The level of feeding had no effect on mean portal concentration of GRF (restricted: 5.5 +/- 0.8, normal: 6.6 +/- 1.4 pg/ml), GRF pulse amplitude (14.7 +/- 2.3 vs. 13.5 +/- 0.7 pg/ml), or GRF pulse frequency (5.3 +/- 1.1 vs. 6.7 +/- 0.9 pulses/8 h). Portal concentrations of SRIF in sheep on a restricted diet were half (P less than 0.01) those of sheep fed a normal diet (10.2 +/- 2.3 vs. 19.6 +/- 1.6 pg/ml). Pulses of SRIF were not significantly associated with changes in GH or GRF concentrations. These data indicate a functional role for hypothalamic GRF in initiating GH pulses. Furthermore, the increase in GH secretion in underfed sheep was most probably due to a decrease in the release of SRIF into hypophysial portal blood. Restricted feeding had no affect on GRF secretion, but because of the reduced exposure of the pituitary gland to SRIF, it is possible that responsiveness to GRF is enhanced.     

外源性牛初乳短链IGF-1治疗改善糖尿病大鼠GH/IGF-1轴的紊乱

目的　观察外源性牛初乳短链胰岛素样生长因子 - 1(BC- t IGF- 1)对糖尿病生长激素 GH/ IGF- 1轴的影响。方法　链脲佐菌素 (STZ) 5 5 mg/ kg空腹腹腔注射 (IP)制备糖尿病大鼠模型 ,随机分为糖尿病对照组 (DM- C,生理盐水0 .1ml/ d IP)和糖尿病治疗组 (DM- BC,BC- t IGF- 12 5 0 ng/ kg· d- 1 IP)。并设正常对照组 (NC) ,每组 n=5。治疗前测定尾尖血血糖 (BG0 ) ,治疗 6周后测定尾尖血血糖 (BG)、血清果糖胺 (FMN)、甘油三酯 (TG)、胆固醇 (CHO)、GH、IGF- 1和肝脏 IGF- 1含量。结果　与 NC相比 ,DM- C血清 FMN、TG明显升高同时血清 GH升高而血清和肝脏 IGF- 1降低 ;与DM- C相比 ,DM- BC血清 FMN、TG明显降低同时血清 GH降低而肝脏 IGF- 1显著升高。结论　外源性 BC- t IGF- 1治疗改善糖尿病大鼠代谢同时改善了糖尿病时的 GH/ IGF- 1轴的紊乱     

GH substitution reverses the growth phenotype but not the defective ossification in thyroid hormone receptor alpha 1-/-beta-/- mice

Thyroid hormone receptor alpha 1, beta 1 and beta 2-deficient mice (TR alpha 1-/-beta-/- mice) demonstrate growth retardation and defective ossification in the epiphyses associated with an inhibition of the GH/IGF-I axis. There are differences between TR alpha 1-/-beta-/- mice (receptor deficient) and the hypothyroid animal model (ligand deficient). Such differences include possible repressive actions exerted by unliganded receptors in the ligand-deficient (hypothyroid) model but not in the receptor-deficient model. In the present study we have investigated whether or not GH substitution rescues the skeletal phenotype of TR alpha 1-/-beta-/- mice. TR alpha 1-/-beta-/- and wild-type (WT) mice were treated with GH from day 18 until 10 weeks of age. GH substitution of mutant mice resulted in a significant and sustained stimulatory effect on the body weight that was not seen in WT mice. GH-treated mutant mice but not GH-treated WT mice demonstrated increased length and periosteal circumference of the femur. However, GH substitution did not reverse the defective ossification seen in TR alpha 1-/-beta-/- mice. TR alpha 1-/-beta-/- mice displayed increased width of the proximal tibial growth plate, which was caused by increased width of the proliferative but not the hypertrophic layer. GH substitution did not restore the disturbed morphology of the growth plate in TR alpha 1-/-beta-/- mice. In summary, GH substitution reverses the growth phenotype but not the defective ossification in TR alpha 1-/-beta-/- mice. Our data suggest that TRs are of importance both for the regulation of the GH/IGF-I axis and for direct effects on cartilage.